The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase.

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.

Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae.

We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake. S. cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells. A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities. The predicted FRE1 protein appears to be a membrane protein and shows homology to the beta-subunit of the human respiratory burst oxidase. These data suggest that FRE1 is a structural component of the ferric reductase. Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S. cerevisiae ferric reductase. An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes. Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.

Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron.

The principal iron uptake system of Saccharomyces cerevisiae utilizes a reductase activity that acts on ferric iron chelates external to the cell. The FRE1 gene product is required for this activity. The deduced amino acid sequence of the FRE1 protein exhibits hydrophobic regions compatible with transmembrane domains and has significant similarity to the sequence of the plasma membrane cytochrome b558 (the X-CGD protein), a critical component of a human phagocyte oxidoreductase, suggesting that FRE1 is a structural component of the yeast ferric reductase. FRE1 mRNA levels are repressed by iron. Fusion of 977 base pairs of FRE1 DNA upstream from the translation start site of an Escherichia coli lacZ reporter gene confers iron-dependent regulation on expression of beta-galactosidase in yeast. An 85-base-pair segment of FRE1 5' noncoding sequence contains a RAP1 binding site and a repeated sequence, TTTTTGCTCAYC; this segment is sufficient to confer iron-repressible transcriptional activity on heterologous downstream promoter elements.

Sp1 represses IL-2 receptor alpha chain gene expression.

Sp1 is a DNA-binding protein that acts as a positive regulator of eukaryotic gene expression. The interleukin-2 receptor alpha chain (IL2R alpha) gene 5' regulatory region contains a single Sp1 consensus motif that overlaps a CArG box capable of binding serum response factor (SRF). The CArG box has previously been shown to be important for IL2R alpha gene expression. In this study, the results of competition experiments suggest that Sp1 and SRF compete for binding to the CArG region. Site-directed mutagenesis and transient transfection assays indicate that the IL2R alpha gene Sp1 serves the unusual role of repressing gene expression, most likely by competing for binding of nuclear factor(s) to the CArG box.

The same target sequences are differentially important for activation of the interleukin 2 receptor alpha-chain gene in two distinct T-cell lines.

High-affinity receptors for interleukin 2 (IL-2) are expressed on T cells following activation. These receptors are composed of both alpha and beta chains. Expression of alpha chains and, therefore, expression of high-affinity receptors are critically regulated at the level of transcription initiation. We have further dissected the regulatory elements involved in controlling transcription of the IL-2 receptor alpha-chain (IL-2R alpha) gene. The IL-2R alpha promoter contains a kappa B site and binding sites for additional nuclear factors within a 50-base-pair region (positions -290 to -240 relative to the major transcription start site). These include one upstream of the kappa B site and one similar to the c-fos serum response element (SRE), which is downstream of the kappa B site. Mutation of the kappa B site decreases IL-2R alpha promoter activity in MT-2 cells (a T-cell line that has been transformed with human T-cell lymphotropic virus type I), but not in Jurkat cells (a T-cell leukemia line) that have been activated by phorbol 12-myristate 13-acetate (PMA). In contrast, mutation of a region upstream of the kappa B site decreases activity in PMA-induced Jurkat cells but increases activity in MT-2 cells. Mutation of the SRE-like site decreases activity in both cell types but the effect in PMA-induced Jurkat is more pronounced. Thus, these distinct cis-acting elements play different physiological roles in IL-2R alpha gene activation in MT-2 cells and PMA-induced Jurkat T cells. These studies provide direct evidence for a functionally significant SRE-like sequence in a gene other than c-fos and the actin genes and identify other elements that are critical for IL-2R alpha gene expression.

Delineation of an enhancerlike positive regulatory element in the interleukin-2 receptor alpha-chain gene.

We have delineated a positive regulatory element in the interleukin-2 receptor alpha-chain gene (IL-2R alpha) between positions -299 and -243 that can potently activate a heterologous (herpesvirus thymidine kinase [tk]) promoter in phorbol myristate acetate (PMA)-induced Jurkat T cells and is functional when cloned in either orientation. This enhancerlike element contains a site (-268/-257) that can bind NF-kappa B; however, unlike the immunoglobulin kappa gene kappa B enhancer element, the IL-2R alpha kappa B-like site alone can only weakly activate a heterologous promoter. Adjacent 5' and 3' sequences also weakly activate the tk-CAT vector, but constructs combining the IL-2R alpha kappa B-like site plus adjacent 5' and 3' sequences potently activate gene expression. This combination of regions is essential for potent PMA-induced transcription from the tk promoter. Experiments using constructs in which IL-2R alpha upstream sequences are sequentially deleted suggested that there is a region 5' of position -299 which can suppress IL-2R alpha promoter and/or enhancer activity. Thus, it is possible that both positive and negative elements may be important in the regulation of IL-2R alpha gene transcription.

The complete nucleotide sequence of canine brain B creatine kinase mRNA: homology in the coding and 3' noncoding regions among species.

To define the structure of canine B creatine kinase, clones were isolated from a library prepared from dog brain mRNA and constructed in the vector lambda gt11. The entire coding portion, the complete 3' nontranslated region, and 43 bp of the 5' noncoding region are reported. Comparison of the predicted amino acid sequence of canine B creatine kinase with the sequence of canine M creatine kinase shows 81% identity. When compared to cDNAS encoding B creatine kinase isolated form other species unusual and striking nucleotide sequence identity in the 3' noncoding region is present. Moreover, two B creatine kinase clones (BCK2 and BCK38) demonstrate microheterogeneity within the 3' nontranslated region indicating variable processing of B creatine kinase pre-mRNA or the existence of multiple genes encoding canine B creatine kinase.

The nature of post-translational formation of MM creatine kinase isoforms.

Isoforms (derived from the same isoenzyme but distinguished by differences in isoelectric point) of MM creatine kinase appear in plasma after myocardial infarction. They are formed by conversion of the tissue form of creatine kinase (MM-A, pI 7.80) to progressively more acidic species (MM-B, pI 7.50) and MM-C (pI 7.20) after release into the circulation. To define the changes responsible for myocardial MM creatine kinase isoform formation in humans and dogs, purified isoforms were treated with trypsin or cyanogen bromide. The digests were fractionated by reverse-phase high pressure liquid chromatography. Comparison of proteolytic maps showed that MM-A and MM-C were each characterized by a single, unique peptide peak. Maps of MM-B creatine kinase contained both of these peaks. Sequence analysis and comparison with the complete amino acid sequence of MM creatine kinase showed that the peptide unique to MM-A corresponded to the COOH-terminal tryptic or CNBr peptide. The peptide unique to MM-C was shown to have the same amino acid composition except for lysine (the COOH-terminal amino acid). Thus, isoform formation is characterized by the successive removal of the COOH-terminal lysine residue from one M subunit at a time resulting in the conversion of MM-A to isoforms MM-B and MM-C.